Fig 2: Colocalization of Arid1a and Gli1+ cells and their progeny in developing roots(A–H) H&E staining (A–D) and Arid1a immunofluorescence (red) (E–H) of the first mandibular molar of a wild-type (WT) mouse from PN4.5 to PN21.5. Mes, dental mesenchyme; epi, dental epithelium. (I–L) Co-immunofluorescence of Arid1a (green) and Gli1 (stained by ß-gal in red) in the first mandibular molar of a Gli1-LacZ mouse at PN4.5. Box in (I) is shown at higher magnification at the right. Arrows indicate positive signals. White dashed lines outline Hertwig’s epithelial root sheath (HERS).(M–P) Arid1a immunofluorescence (green) and visualization of tdTomato (red) of the first mandibular molar of a Gli1-CreER;tdTomato mouse at PN18.5 after induction at PN3.5. The progeny of the Gli1+ lineage show red signal. Arrows indicate positive signals. TdT, tdTomato; DPC, dental pulp cell; OD, odontoblast; PDL, periodontal ligament. Schematic at the bottom indicates induction protocol. TAM, tamoxifen. Scale bars: 100 µm.

Fig 3: Loss of Arid1a in Gli1+ root progenitor cells leads to downregulated Hh signaling activity(A) Heatmap hierarchical clustering showing the gene expression profiles in the apical regions of control and Gli1-CreER;Arid1afl/fl mouse molars at PN7.5.(B–M) RNAscope in situ hybridization (red) of Gli1 (B–E), Ptch1 (F–I), and immunofluorescence of Ccnd1 (red, J–M) of first mandibular molars of control and Gli1-CreER;Arid1afl/fl mice at PN7.5. The boxed areas are enlarged on the right. Arrows indicate positive signals in control group; arrowheads indicate reduced signal in targeted region of Gli1-CreER;Arid1afl/fl mouse molars.Schematic at the bottom indicates induction protocol. Scale bars: 100 µm. See also Figures S4 and S5.

Fig 4: Arid1a interacts with Plagl1 to regulate Gli1 transcription during tooth root development(A) Chromatin immunoprecipitation (ChIP) assay with Arid1a antibody (or immunoglobulin G [IgG]), followed by qPCR with two pairs of primers. n = 3, *p < 0.05.(B) The Plagl1 motif identified based on the motif enrichment analysis on WT-specific ATAC-seq peaks.(C) RNAscope in situ hybridization of Plagl1 (green) and Gli1 (red) of first mandibular molars of control mice at PN7.5.(D) The Plagl1 motif hits are found on ATAC regions at the intron 2 of Gli1 and promoter region of Ptch1 and Ccnd1.(E) CoIP assay with Arid1a antibody (or IgG), followed by immunoblotting of Arid1a and Plagl1.(F) Western blot of Gli1 in cultured DPCs treated with control siRNA or Plagl1 siRNA.(G) RNAscope in situ hybridization of Dspp (green) and the western blot of Dspp in cultured DPCs treated with control siRNA or Plagl1 siRNA after 6 days of odontogenic induction.Data are represented as mean ± SD. Scale bars: 100 µm. See also Figure S7.

Fig 5: An OV targeting PD-L1 via extracellular vesicle delivery of shPD-L1 molecules has enhanced therapeutic activity in vivo.a Immunoblot analysis showing protein expression levels of PD-L1, GAPDH (loading control), and total proteins in B16-F10 cells subjected or not to VSV?51-amiR-NTC or VSV?51-shPD-L1 infection (n = 3). b, c Schematic representation (b) of transwell coculture assays designed to assess the transfer of shPD-L1 via infected cell-derived SEVs to uninfected cells. c Immunoblot analysis of PD-L1, GAPDH and VSV protein levels in producer cells (upper compartment) and receiving cells (lower compartment) after 48 h of education by cell-secreted factors derived from VSV?G-shPD-L1-infected cells (upper compartment). Both uninfected and VSV?51-infected cell lysates are included as controls. Immunoblots are representative of three biological replicates. d Kaplan–Meier survival curves of mice bearing subcutaneous (SC) B16-F10 tumours and treated as indicated with vehicle control (PBS) or with VSV?51-amiR-NTC control or VSV?51-shPD-L1 (n = 10 mice per group). Dotted vertical lines indicate virus treatments. Log-rank (Mantel–Cox) test, ****P < 0.0001. e Mice that had completely cleared B16-F10 tumours upon VSV?51-shPD-L1 treatment (in d) were re-challenged SC with 5 × 105 B16-F10 cells on day 93 (n = 4). Arrows indicate initial VSV?51-shPD-L1 treatments. f B16-F10 and TH04 cells were infected with indicated OVs (VSV?51-amiR-NTC, VSV?51-shPD-L1, VSV?51-amiR-4, VSV?51-amiR-4-shPD-L1) at MOIs of 0.01 and 0.001, respectively. Cell lysates were collected 48 hpi and prepared for immunoblotting with antibodies against ARID1A, PD-L1, and ß-actin (loading control), n = 3. g Schematic showing the bystander effects of VSV?51-amiR-4-shPD-L1. In our model, cell death occurs in a two-pronged attack against cancer cells; following OV infection of cancer cells and via synthetic lethal interactions in target cells receiving ARID1A-targeting amiR-4 delivered by SEVs from neighbouring OV-infected cells and systemic administration of the EZH2 methyltransferase inhibitor GSK126. In addition, another bystander effect is induced by shPD-L1-containing SEVs derived from VSV?51-shPD-L1-infected cells. While OV infection induces PD-L1 protein levels, expression of an shRNA targeting PD-L1 from an OV backbone decreases PD-L1 to baseline levels and enhances T cell-mediated death of cancer cells. Source data are provided as a Source data file.